Cα gain-of-function variant in Alzheimer’s disease displays enhanced catalysis by a mechanism that evades down-regulation

Julia A. Callendera,b, Yimin Yanga, Gema Lordéna, Natalie L. Stephensonc, Alexander C. Jonesb, John Brognardc,d, and Alexandra C. Newtona,1

aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; bBiomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093; cCancer Research UK Manchester Institute, The University of Manchester, Manchester 4BX M20, United Kingdom; and dLaboratory of Cell and Developmental Signaling, National Cancer Institute at Frederick, Frederick, MD 21702

Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved May 9, 2018 (received for review March 28, 2018) Conventional C (PKC) family members are reversibly We recently identified gain-of-function mutations in PKCα in + activated by binding to the second messengers Ca2 and diacylgly- Alzheimer’s disease (AD) (23), a degenerative pathology char- cerol, events that break autoinhibitory constraints to allow the acterized by the accumulation of amyloid-β (Aβ)proteinaggre- to adopt an active, but degradation-sensitive, conforma- gates in the brain and synaptic degeneration (24). Whole-genome tion. Perturbing these autoinhibitory constraints, resulting in pro- sequencing of individuals from 410 families with late-onset AD tein destabilization, is one of many mechanisms by which PKC function identified germline PKCα mutations in affected, but not in is lost in cancer. Here, we address how a gain-of-function germline disease-free, individuals in six of the families. These mutations all α ’ mutation in PKC in Alzheimer s disease (AD) enhances signaling with- enhanced the agonist-evoked signaling output of PKCα assessed out increasing vulnerability to down-regulation. Biochemical analyses using a genetically-encoded PKC activity sensor in overexpression of purified protein demonstrate that this mutation results in an ∼30% studies (23). While the mechanisms by which Aβ aggregation leads increase in the catalytic rate of the activated enzyme, with no changes + to synaptic degeneration are not fully understood, electrophysio- in the concentrations of Ca2 or lipid required for half-maximal activa- logical studies established that the synaptic effects of Aβ depend BIOCHEMISTRY tion. Molecular dynamics simulations reveal that this mutation has α α’ both localized and allosteric effects, most notably decreasing the dy- on PKC via a mechanism requiring PKC s PDZ ligand [which namics of the C-helix, a key determinant in the catalytic turnover of scaffolds to PDZ domain-containing such as PSD95, . Consistent with this mutation not altering autoinhibitory con- SAP97, and PICK1 (25, 26)] and its interaction with the scaffold straints, live-cell imaging studies reveal that the basal signaling output PICK1 (23, 27). Thus, enhanced activity of PKCα may augment of PKCα-M489V is unchanged. However, the mutant enzyme in cells the degenerative pathways activated by Aβ. Supporting a general displays increased sensitivity to an inhibitor that is ineffective toward role of enhanced PKC activity in AD, a recent unbiased phospho- scaffolded PKC, suggesting the altered dynamics of the kinase domain proteomics study identified increased of PKC may influence protein interactions. Finally, we show that phosphor- substrates, including myristoylated alanine-rich C-kinase substrate ylation of a key PKC substrate, myristoylated alanine-rich C-kinase (MARCKS), as a primary early event in AD (28). These activity- substrate, is increased in brains of CRISPR-Cas9 genome-edited mice enhancing mutations are in stark contrast to loss-of-function cancer- containing the PKCα-M489V mutation. Our results unveil how an associated mutations. Indeed, with the possible exception of a AD-associated mutation in PKCα permits enhanced agonist- mutation in PKCβ in adult T-cell leukemia (29), it is noteworthy dependent signaling via a mechanism that evades the cell’shomeo- α static down-regulation of constitutively active PKC . Significance

protein kinase C | PKC | Alzheimer’s disease | | ’ enzyme mutation This work unveils how an Alzheimer s disease-associated muta- tion (M489V) in protein kinase Cα (PKCα) enhances catalytic ac- ’ α α tivity without sensitizing the protein to the cell s homeostatic rotein kinase C (PKC ) is a ubiquitously expressed member degradation of aberrantly active PKCα. The active conformation Pof the protein kinase C (PKC) family of / of wild-type PKC is sensitive to degradation, and therefore kinases that transduces signals mediated by the second messengers 2+ constitutively activated PKC paradoxically manifests as loss of Ca and diacylglycerol (DG) (1, 2). It plays a major role in sup- function. We show that PKCα-M489V enhances the intrinsic – pressing cell proliferation (3 7) and survival (8, 9). Studies both in catalytic rate of the kinase without altering the equilibrium be- mice, where its deletion causes the spontaneous development of tween the autoinhibited (protected) conformation and the acti- colon tumors (10), and in patients, where loss of protein expression vated (degradation-sensitive) conformation. Thus, the on/off is associated with colon cancer (11), have long supported a role as a dynamics are unchanged, but reactions are catalyzed at a faster tumor suppressor. It is the first PKC in which a cancer-associated rate when the enzyme is on. These findings are significant be- mutation was reported: Over 20 y ago, Joubert and coworkers cause they provide a mechanism through which a disease mu- reported that a mutation in PKCα in human pituitary tumors tation in PKCα causes aberrant activation without resulting in resulted in mislocalization, thus allowing growth in soft agar in paradoxical loss of function via degradation. ectoptic expression studies (12–14). Loss-of-function somatic mu- tations in PKCα have now been identified in many diverse cancers Author contributions: J.A.C., Y.Y., G.L., N.L.S., J.B., and A.C.N. designed research; J.A.C., (15). Mounting evidence suggests that PKCα opposes oncogenic Y.Y., G.L., N.L.S., and A.C.J. performed research; J.A.C., Y.Y., G.L., N.L.S., A.C.J., J.B., and survival signaling by its phosphorylation and inactivation of onco- A.C.N. analyzed data; and J.A.C. and A.C.N. wrote the paper. genes such as K-Ras (8, 16), growth factor receptors (17–19), and The authors declare no conflict of interest. phosphatidylinositol-3 kinase (20–22), among others. The key role This article is a PNAS Direct Submission. of PKCα in suppressing proliferative and survival pathways poise it Published under the PNAS license. to play a role not only in cancer via its loss of function but also in 1To whom correspondence should be addressed. Email: [email protected]. degenerative diseases via gain of function.

www.pnas.org/cgi/doi/10.1073/pnas.1805046115 PNAS Latest Articles | 1of9 Downloaded by guest on September 24, 2021 that no gain-of-function mutations in any of the nine PKC Here, we examine the mechanism by which one AD-associated have been identified (15). variant, PKCα-M489V, enhances PKCα signaling. This variant PKCα is an exquisitely tuned enzyme whose signaling output was identified in seven individuals in four families: It is hetero- depends not only on binding to second messengers but also on the zygous, with one allele encoding a protein with a Met-to-Val abundance of inputs that control its steady-state levels in the cell. substitution at position 489 in the activation loop, a segment Following its biosynthesis, PKCα becomes constitutively phos- near the entrance to the that critically controls phorylated at three priming sites, allowing it to adopt an auto- catalysis (33). Our findings support a model in which altered inhibited conformation in the cytosol that protects the enzyme from dynamics of the kinase domain enhance the activity of the PKCα- dephosphorylation and degradation (1). Because this autoinhibited M489V variant by increasing the intrinsic catalytic rate of the speciesisresistanttodegradation,PKCα is a particularly long-lived enzyme without affecting autoinhibitory constraints. Thus, the ’ enzyme. It is transiently and reversibly activated by signals that enzyme s on/off dynamics are unaffected, but when on it cata- promote hydrolysis to generate its two second mes- lyzes phosphorylation at a faster rate. This provides a previously + + sengers, Ca2 and DG: Ca2 binds the C2 domain, promoting as- undescribed mechanism by which a disease-associated mutation + sociation to the plasma membrane via a Ca2 -dependent bridge enhances PKC activity without compromising its stability. with anionic , and DG binds one of two tandem C1 Results domains (30). Engagement of both membrane-targeting modules PKCα-M489V Mutation Confers an Enhanced Catalytic Rate. We pre- with the plasma membrane results in release of an autoinhibitory viously established that PKCα-M489V displays enhanced agonist- pseudosubstrate segment from the substrate-binding cavity, po- α “ ” evoked signaling output relative to wild-type PKC in cells (23). sitioning PKC in an open and signaling-competent conforma- Specifically, the M489V variant overexpressed in COS7 cells has tion. Metabolism of DG results in PKC disengaging from the ∼25% greater activity compared with wild-type enzyme following + membrane and regaining the stable, autoinhibited conformation agonist stimulation of cells to generate Ca2 and DG, as assessed (30). Phorbol esters, potent competitive ligands for DG binding, using the genetically-encoded FRET-based C kinase activity re- are not readily metabolized and therefore result in irreversible porter (CKAR) (34, 35). This variant has a replacement of Met to binding of PKC to the membrane, leading to acute activation but Val at position 489 in the activation loop, nine residues before the chronic down-regulation of PKC protein levels (31, 32). The constitutive phosphorylation site (Thr497) for the upstream kinase paradoxical effect of these potent tumor promoters—initial ac- PDK-1 (Fig. 1A). It is also two residues removed from an in- tivation followed by eventual degradation—confounded un- teraction network that connects the activation loop to the C helix, derstanding the role of PKC in cancer (9). Given that the active in turn connecting to the C-terminal phosphorylation site Ser657 conformation of PKC is sensitive to degradation, how do AD- (Fig. 1C). Furthermore, this face of the kinase domain for the associated mutations result in enhanced cellular kinase activity related PKCβII has been shown to interface with the C2 domain while also evading down-regulation? to maintain the enzyme in an autoinhibited conformation (36), as

A B C1A pseudosubstrate PDZ ligand C1B C2 C1A C1B C2 kinase C-tail C-tail NCP P P hinge M489 kinase pseudosubstrate C pS657 G655

R462 K388 K486 R389 pT497(E) E487 M489

T495 V493

Fig. 1. AD-associated PKCα mutation in key region of catalytic domain. (A) Primary structure of PKCα showing pseudosubstrate (red), C1A and C1B domains (orange), C2 domain (yellow), kinase domain (cyan), C-terminal tail (gray), and PDZ ligand (purple). Indicated are the three processing phosphorylation sites, Thr497 (magenta circle) in the activation loop and Thr638 (orange circle) and Ser657 (green circle) in the C-terminal tail. (B) Cartoon representation showing autoinhibitory constraints in the full-length inactive form of PKCα wherein the pseudosubstrate occupies the substrate binding cavity, an interaction locked in place by the C2 domain. (C) Structure of PKCα kinase domain (PDB ID code 3IW4) showing position of Met489 (red space filling) in the activation loop; note this structure has a Glu at the position of the activation loop phosphorylation site [pT497(E)]. Indicated in stick representation is the network of interactions from the activation loop to the C-terminal tail that involves a salt bridge from Glu487 in the activation loop to Arg389 in the C-helix and H-bonding between the Lys388 in the C-helix to the main chain of Gly655 in the C-terminal tail; H-bonds and salt bridges are indicated by yellow dots. Gly655 is in a conserved FXXF motif that directly precedes the hydrophobic phosphorylation site (phospho-Ser657); the two Phe are indicated in gray space filling model. Residues that pack into the activation loop (Arg462, Lys486, Val493, and Thr495) are also presented in gray space filling model.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1805046115 Callender et al. Downloaded by guest on September 24, 2021 schematized in Fig. 1B. Thus, this residue is positioned to po- celles afford a highly sensitive system to dissect the lipid de- tentially perturb autoinhibitory constraints, catalysis, or both. pendence of PKC activation, which displays high To elucidate the molecular mechanism through which the with respect to PS (37–39). Kinase assays revealed no differences + M489V mutation confers enhanced PKC activity in cells, we in the two proteins with respect to the concentration of Ca2 or analyzed the kinetics of activation of the purified wild-type and PS resulting in half-maximal activity (Fig. 2B and Table 1), nor M489V mutant protein. GST-tagged PKCα was purified to ho- was the basal activity different between the two proteins, which mogeneity from insect cells using a baculovirus expression sys- both displayed ∼10% of -induced activity when activa- tem. Western blot analysis confirmed that both the wild-type and tors were absent from the assay. We did note, however, that the α PKC -M489V proteins were processed by priming phosphory- Vmax was consistently higher for the PKCα-M489V protein lations at the activation loop (Thr497) and the two C-terminal compared with wild-type enzyme. These data reveal that while sites (Thr638 and Ser657) (Fig. 2A). the autoinhibitory constraints were unaffected in the PKCα- To assess whether autoinhibitory constraints were altered in M489V variant, the maximal velocity was higher. the PKCα-M489V protein, we examined the dependence for We next assessed whether peptide substrate or ATP depen- + activation on (i)Ca2 or (ii) mole fraction dencies were altered in the AD variant. When assayed in the 2+ (PS) in DG-containing micelles. Triton X-100 lipid mixed mi- presence of saturating Ca and lipid, the Km for peptide substrate

A

-PKC -M489V T GST GST-M489V GST-PKCGS PKC 250 100 150

pT497 BIOCHEMISTRY 100 100 75 pT638 100 50 pS657 37 100

B PKC PKC -M489V ) -1 Activity (units mg PKC

[free Ca2+] (µM) [PS] (mol %) ) -1 Activity (units mg PKC

[peptide substrate] (µM) [ATP] (µM)

Fig. 2. Purified PKCα-M489V displays higher Vmax than wild-type PKCα under activating conditions. (A) Coomassie Blue-stained SDS/PAGE gel of purified GST- PKCα wild type and GST-PKCα M489V (Left). Western blot of pure proteins probed with antibodies for total PKCα, or with phosphospecific antibodies to the priming phosphorylation sites, pT497, pT638, and pS657 (Right). (B) The activity of PKCα wild type (blue) or M489V (red) (typically 2.4 nM) measured as a + function of Ca2 , PS, peptide substrate, or ATP concentration. Data are graphed in units (nanomoles phosphate per minute) per milligram GST-PKC. Data represent the average ± SEM of triplicate samples.

Callender et al. PNAS Latest Articles | 3of9 Downloaded by guest on September 24, 2021 Table 1. Kinetic values for in vitro kinase assays using purified PKCα or PKCα-M489V 2+ −1 Protein Ca 50, μMPS50, mol % Kmsubstrate, μM KmATP, μM Vmax, units·mg

PKCα 0.17 ± 0.05 6.16 ± 0.05 24 ± 555± 8(2.0± 0.2) × 103 PKCα-M489V 0.21 ± 0.07 6.16 ± 0.06 28 ± 447± 6(2.7± 0.2) × 103

Kinetic values from the data shown in Fig. 2B are listed. Data represent the average ± SEM of triplicate samples. Unless otherwise indicated in the figure legends, the standard reaction component concentrations were as + follows: 200 μM free Ca2 , Triton X-100 mixed micelles containing 5 mol % DG and 15 mol % phosphatidylserine,

100 μM peptide substrate, and 100 μM ATP. Vmax is obtained from the experiment varying ATP concentration (all other components are as noted for the standard reaction). Vmax is reported as units per milligram GST-PKC, where one unit is defined as 1 nmol phosphate hydrolyzed per minute.

and the Km for ATP were indistinguishable from wild-type enzyme that stabilizes the active conformation of kinases. For PKCα, (Fig. 2B and Table 1). However, the Vmax was higher for PKCα- Arg389 in the C-helix forms an electrostatic interaction with Glu487 3 M489V [Vmax = (2.7 ± 0.2) × 10 nmol phosphate per minute per in the activation loop, two residues removed from Met489, whereas 3 mg PKC] compared with wild type [Vmax = (2.0 ± 0.2) × 10 nmol the flanking Lys388 forms an H-bond with the main chain carbonyl of phosphate per minute per mg PKC], an increase observed in each Gly655, part of a conserved FXXF motif (33) and two residues re- of five separate protein preparations. Analysis of protein obtained moved from phosho-Ser657, a key determinant of stability of PKCα’s from three separate preparations, under conditions of saturating kinase domain (Fig. 1C). Additionally, the simulations indicated that + Ca2 and lipid, 100 μM peptide, and 300 μM ATP, revealed a 29 ± motions in the distal αGandαI helices were increased in the M489V 9% increase in the specific activity of purified PKCα-M489V mutant compared with wild-type PKCα. These studies support a compared with wild type. These data reveal that neither the de- model in which replacing Met with Val at position 489 affects the gree of autoinhibition nor the cofactor-dependent release of dynamics not only around the active site but also at distal surfaces on autoinhibition is altered by the M489V mutation. Rather, Met-to- the kinase domain. Val substitution increases the intrinsic catalytic rate of the enzyme. PKCα-M489V Is Not More Basally Active than Wild-Type PKCα in a Molecular Dynamics Simulations Reveal Altered Dynamics of the Catalytic Cellular Environment. Given that the M489V mutation does not Domain in PKCα-M489V. To gain insight into how the M489V muta- affect autoinhibitory constraints in the pure protein, we hy- tion increases the catalytic rate without altering sensitivity to co- pothesized that basal (unstimulated) signaling by PKCα-M489V factors, binding of peptide substrate, or the Km for ATP, we would be similar to that of wild-type PKCα in cells. We over- compared the dynamics of the kinase domain of wild type and expressed a FRET-based reporter for PKC activity (CKAR, ref. PKCα-M489V using molecular dynamics simulations (Fig. 3). These 34) alone or with either wild type or mutant mCherry-PKCα in simulations reveal both localized and long-range changes in the COS7 cells and monitored the decrease in phosphorylation of dynamics of the kinase core. Notably, altered dynamics were ob- CKAR, assessed by the decrease in FRET ratio, following ad- served in two key segments of the ATP binding pocket: (i)Motion dition of the general PKC inhibitor bisindolylmaleimide IV (Bis was increased in the Gly-rich loop, a key determinant between the IV) (Fig. 4A). Bis IV caused a drop in the FRET ratio of cells βIandβII strands (β1/βII) that anchors the β-phosphate of ATP transfected with CKAR alone, representing inhibition of the (33), and (ii) motion was decreased in the C-helix (αB/αC), a segment basal activity of endogenous PKC isozymes (open triangles). This

A PKC PKC -M489V

0.35 1/ II B/ C activation loop (AL) G I 0.30 0.25 0.20 0.15 0.10 RMSF (nm) 0.05 0.00 325 350 375 400 425 450 475 500 525 550 575 600 Residue Number B PKC M489V II

1 B Gly loop AL C

G Met489 I Val489

Fig. 3. M489V mutation causes key alterations in the dynamics of the kinase domain structure. (A) Molecular dynamics simulations showing altered flexibility around the ATP , αG helix, and αI helix of the M489V variant. RMSF of each residue are shown (A) graphically and (B) structurally, with width and color of the ribbon corresponding to the level of fluctuations observed. Key regions are highlighted as is the residue at position 489.

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1805046115 Callender et al. Downloaded by guest on September 24, 2021 n.s.

A BisIV (2 µM) B BisIV (2 µM) C 1.00 1.00 1500 CKAR CKAR 0.95 PKC 0.95 n.s. PKC 1000 0.90 0.90 500 (mCherry, r.u.) (mCherry,

0.85 0.85 PKC Expression

PKC Activity (r.u.) 0.80 PKC Activity (r.u.) 0.80 0 10 20 10 20

Time (min) Time (min) PKC

PKC

Fig. 4. Basal activity of wild type and PKCα-M489V is the same, but removal of regulatory moiety unmasks enhanced activity of the M489V kinase domain. (A) COS7 cells overexpressing CKAR alone (open triangles) or coexpressing CKAR and full-length mCherry-PKCα wild type (blue circles) or M489V (red squares) were exposed to 2 μM Bis IV and the change in FRET monitored (n ≥ 20 from at least three separate biological replicates). (B) COS7 cells overexpressing CKAR alone (open triangles) or coexpressing CKAR and the catalytic domain of PKCα (αCat) wild type (open blue circles) or M489V (open red squares). Two mi- cromolar Bis IV was added and the change in FRET was monitored. (n ≥ 54 from at least three separate biological replicates). For both A and B, data were normalized to the first 5 min before Bis IV addition and are graphed as average ± SEM. Note that in some cases the error bars are obscured by the symbols. (C) Quantification of average mCherry-PKC expression for each overexpressed PKC depicted in A and B. Data are graphed as average mCherry intensity ± SEM (n.s., not significantly different using a Student’s t test).

drop was larger in cells overexpressing PKCα (blue), with the M489V has enhanced sensitivity to the ATP-competitive inhibitor BIOCHEMISTRY difference reflecting the basal activity of the overexpressed (Gö6976) that discriminates between scaffolded and nonscaffolded PKCα. Importantly, Bis IV caused the same drop in cells over- PKC, but not to the nondiscriminatory inhibitor (Bis IV). expressing comparable levels of PKCα-M489V (red). Thus, the To determine whether the altered inhibitor sensitivity reflected M489V mutation does not enhance the basal signaling output of an intrinsic property of the kinase or altered cellular interactions, PKCα; enhanced signaling is only observed following agonist we measured the Ki of purified GST-PKCα for Gö6976: The Ki of stimulation and removal of autoinhibitory constraints (23). M489V (109 ± 20 nM) and wild-type enzyme (113 ± 10 nM) were To assess if the M489V mutation confers increased basal sig- the same within the error of the assay (Fig. 5C). These data sug- naling in the absence of autoinhibitory constraints, we introduced gest that the enhanced sensitivity of the M489V variant to Gö6976 this mutation into a PKCα construct that lacked its N-terminal results from altered interactions with the cellular environment, regulatory moiety (thus lacking the pseudosubstrate, C1 domains, rather than from an innate property of the enzyme itself. and C2 domain). We then measured the effect of inhibiting PKC α activity in cells coexpressing the isolated catalytic domains ( Cat) Phosphorylation of MARCKS Is Increased in the Brains of PKCα-M489V and CKAR (Fig. 4B). Addition of Bis IV produced a significantly Mice. The M489V mutation was identified in affected, but not higher drop in FRET for cells expressing the isolated PKCα cat- unaffected, siblings in four unrelated families, suggesting that it alytic domain containing the M489V mutation (red) compared with the wild-type PKCα catalytic domain (blue). For these ex- is causal in the disease (23). Because increased phosphorylation periments, cells expressing comparable amounts of each kinase of MARCKS has been identified as one of the earliest and most domain were selected for imaging: Fig. 4C depicts the average robust phosphorylation changes in postmortem brains from AD mCherry-PKCα intensity for the experiments in Fig. 4 A and B. patients compared with controls (28), we assessed whether the Thus, the findings in Fig. 4 A and B do not result from differ- AD-associated single change altered phosphorylation ences in the amount of overexpressed protein. YFP expression of MARCKS in an animal model. To this end, we used CRISPR- was used to confirm that all the experiments contained saturating Cas9–mediated genome editing to develop a genetically engi- levels of CKAR. neered mouse containing a homozygous M489V mutation in its Prkca gene (Taconic Biosciences GmbH developed for Cure PKCα-M489V Confers Enhanced Sensitivity to ATP-Competitive Inhibitors Alzheimer’s Fund). We obtained whole-brain lysates from either in Cells. We next took advantage of pharmacological tools to address wild-type or homozygous M489V mice and analyzed them via whether the M489V mutation alters signaling on protein scaffolds. SDS/PAGE and Western blotting for a known PKC phosphory- We have previously shown that scaffold-bound PKC is refractory to lation site on MARCKS protein (Fig. 6). The mice containing the ATP-competitive inhibitors such as Gö6976 and Gö6983 but is M489V mutation displayed a 41 ± 20% increase in phosphoryla- fully sensitive to the uncompetitive inhibitor Bis IV (23, 40, 41). tion of MARCKS at Ser-159/163 compared with wild-type mice. We overexpressed both CKAR and mCherry-PKCα wild type or We also blotted for total PKCα levels to assess whether the M489V in COS7 cells stimulated with the PKC activator phorbol α 12,13-dibutyrate (PDBu) to maximally activate all PKC in the cell, M489V mutation affected the PKC stability and protein levels in followed by treatment with subthreshold amounts of the ATP- an endogenous, whole-brain environment. Importantly, the total α competitive inhibitor Gö6976 (Fig. 5A). Cells expressing PKCα- PKC protein levels were not significantly different between the M489V displayed a greater drop in activity compared with wild- wild-type and M489V samples. This establishes that the M489V type PKCα at all three inhibitor concentrations tested. In contrast, mutation both changes PKCα signaling in the brain to enhance the subsaturating amounts of the ATP-uncompetitive inhibitor Bis IV phosphorylation of one of its major downstream targets and also caused an identical drop in the cellular activity of both wild type does so in a manner that does not alter the steady-state levels of and PKCα-M489V (Fig. 5B). These results reveal that PKCα- total PKCα protein.

Callender et al. PNAS Latest Articles | 5of9 Downloaded by guest on September 24, 2021 A PDBu Gö6976 BCPDBu BisIV 1.02 1.02 1.00 1.00 Gö6976 (µM) BisIV (µM) PKCα 0.98 0.98 PKCα-M489V WT 0.06 0.96 M489V 0.96 WT 1.0 M489V 1.0 0.94 WT 0.2 0.94 M489V WT 2.0 0.92 WT 0.6 0.92 M489V M489V 0.5 PKC Activity (r.u.) (% max) PKC Activity (r.u.) 0.90 0.90

0.88 0.88 PKC α Activity 0 10 20 30 10 20 30 0.1 1 10 100 1000 Time (min) Time (min) [Gö6976] (nM)

Fig. 5. PKCα-M489V in cells is more sensitive to Gö6976 inhibition. (A and B) COS7 cells overexpressing mCherry-PKCα wild type or M489V and CKAR were exposed to PDBu (200 nM) followed by subsaturating amounts of (A) Gö6976: 0.06 μM (purple), 0.2 μM (orange), or 0.6 μM (green) or (B) Bis IV: 1 μM (green) or 2 μM (purple). Data were normalized to the maximal FRET after PDBu addition. (n ≥ 14 from three separate biological replicates). Data are depicted as average ± SEM; note that in some cases the error bars are obscured by the data points. (C) Purified GST-PKCα wild-type (blue) or M489V (red) activity in the presence of Gö6976. Data represent the average ± SD of triplicate samples.

Discussion the case for PKCα, then the altered motions in the active site Here we unveil a previously undescribed mechanism by which a may reduce the constraints that limit ADP release, thereby in- disease-associated mutation in a conventional PKC has en- creasing the catalytic rate. It is noteworthy that, like many en- hanced activity without threatening the stability of the protein zymes (47), PKA and PKC are relatively inefficient, with PKA (Fig. 7). Biochemical and in silico approaches, cellular studies, catalyzing on the order of 20 reactions per second (46) and and analysis of brains from genome-edited mice reveal that the PKCα on the order of 3 reactions per second (this study). Thus, AD-associated PKCα-M489V variant has the same on/off dy- namics as wild-type enzyme but catalyzes reactions at a faster rate when on, resulting in a significant increase in phosphoryla- tion of endogenous PKC substrates without causing a reduction in wt / wt PKCα M489V / M489V the levels of PKCα. We show that autoinhibitory constraints are unperturbed by mutation of this residue, with basal sig- p-MARCKS naling both in vitro and in cells unchanged from wild-type PKCα. 75

Rather, this mutation alters the dynamics of the kinase core in a 75 manner that enhances the rate of catalysis by ∼30% while it is MARCKS open and active, accounting for the previously described increase 50 β-actin in agonist-evoked signaling in cells (23). Furthermore, these 37 altered dynamics affect the cellular pharmacology of the PKCα- M489V variant, rendering it more sensitive to an ATP-competitive 75 inhibitor but not to an uncompetitive inhibitor. Finally, we dem- 50 onstrate that brains from mice with this PKCα mutation contain β-actin 37 higher phosphorylation levels of the canonical PKC substrate 11432 57986 101 12 13 14 15 16 17 18 MARCKS. Given this finding, it is of note that a recent unbiased phosphoproteomics study of brains from human AD patients * n.s. revealed that an increase in MARCKS phosphorylation is the major 2.5 3.0 up-regulated pathway in early stages of this disease (28). 2.0 2.0 The altered catalytic rate resulting from mutation of a bulky 1.5 1.0 Met to the smaller Val in a key regulatory segment involved in PKC 1.0 0.5 structuring the catalytic domain’s active site exemplifies how p-MARCKS 0 0 structural dynamics and protein plasticity play a critical role in catalysis. Notably, elegant NMR relaxation studies with wild-type and mutant forms of cis–trans cyclophilin A provide wt / wt wt / wt strong experimental evidence that protein motions limit the turnover rate of the enzyme (42). Consistent with protein dy- M489V / M489V M489V / M489V namics determining the rate of catalysis, our in silico molecular Fig. 6. Phosphorylation of MARCKS is increased in the brains of PKCα- dynamics simulations support a model in which substituting Met M489V mice. Western blot of lysates of whole brain obtained from nine for Val at position 489 alters both localized and distal structural male and female 3-mo-old wild-type mice (lanes 1–4, males; lanes 5–9, fe- flexibility of the catalytic domain. Such allosteric effects are not males) or nine male and female genome-edited mice containing a homo- surprising given the interlinked functional communities that zygous PKCα-M489V mutation (lanes 10–15, males; lanes 16–18, females). populate the kinase core (33, 43). The activation loop, in par- Western blots were probed with antibodies specific to a known PKC phos- α ticular, acts as a hub for these communities. For example, nu- phorylation site on MARCKS (Ser-159/163) or to total PKC (Top). Bands were merous studies with the archetypical kinase, PKA, and with the quantified using densitometry and the phospho-MARCKS signal was nor- malized to total MARCKS signal and PKCα signal was normalized to its Tec family kinases have established that altering residues in the β-actin loading control. Normalized data from the depicted Western blot activation loop region alters the dynamics of the catalytic domain were plotted (Bottom) as average normalized intensity ± SEM (*P < 0.05; n. (44, 45). Mechanistic studies with PKA have revealed that the s., not significantly different using a Student’s t test). Males are indicated in rate-limiting step in catalysis is the release of ADP (46). If this is green squares and females in black circles.

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1805046115 Callender et al. Downloaded by guest on September 24, 2021 A

B

Fig. 7. PKCα-M489V mutation increases PKCα signaling output without causing PKCα degradation. (A) Alignment of the activation loop of conventional PKC isozymes showing conserved regions, including the flanking DFG and APE motifs, (yellow); residues that frequently harbor loss-of-function mutations in cancer (blue asterisk); and the variable residue at the position corresponding to Met489 in PKCα (red). (B, Left) Model depicting normal PKCα signaling, in which the enzyme is in an autoinhibited and stable conformation that is transiently and reversibly activated. (B, Right) Model showing the homeostatic down- BIOCHEMISTRY regulation of PKCα after either chronic activation by phorbol esters (which trap PKC in an open and labile conformation) or as a result of mutations that disrupt autoinhibitory constraints to constitutively activate PKCα signaling. Rather than promoting the constitutively active form of PKCα that would be subsequently degraded, the PKCα-M489V mutation instead enhances the normal signaling output of the enzyme without leading to its degradation (Left). Domains and phosphorylation sites (small circles) are color-coded as in Fig. 1.

minor changes are likely to have significant effects on catalysis. leles of this effectively suppress growth in 3D whereas Interestingly, the residue at position 489 varies among different one allele does not (15). Thus, small changes in the activity of PKC isozymes (Fig. 7A). Whether the residue at this position PKC have large effects on cellular function. Given the multiple generally serves to tune catalytic efficiency unique to each PKC regulatory inputs necessary for PKC activity, PKC can be readily enzyme remains to be established. Indeed, there exists a precedent inactivated by mutations that prevent processing phosphoryla- for residues in this position playing a key role in tuning the catalysis tions, impede second messenger binding, or impair catalysis. In- + of kinases; namely, the large hydrophobic residue at the DFG deed, such loss-of-function mutations are prevalent in cancer (15, 5 position in c-Src has been shown to act as a “hydrophobic latch” 49, 50). However, mechanisms to enhance the activity of PKC are in its control of c-Src activity (48). In contrast, the flanking residues more difficult to envision. Conventional PKC isozymes engage in in PKC contain the highly conserved DFG and APE motifs that are frequently mutated in cancer to result in loss of function. intramolecular autoinhibition to prevent downstream signaling in We have previously established that PKC bound to protein the absence of activating ligands, and disruption of this auto- scaffolds is refractory to inhibition by ATP-competitive inhibitors inhibition by prolonged treatment with potent PKC activators such (40). This is relevant to the cellular pharmacology of PKCα in as phorbol esters and bryostatins causes the degradation of PKC the context of AD because the species of PKCα that mediates (31, 32, 36) (Fig. 7B, Right). Indeed, patients receiving prolonged Aβ’s effects on synaptic transmission depends on PKCα’s PDZ bryostatin infusions in a clinical trial for advanced metastatic ligand, which scaffolds to the PDZ domain-containing proteins cancer were reported to have decreased levels of PKC in pe- PICK1 and PSD95 (25, 26). In this study, we found that PKCα- ripheral blood monocytes (51). Thus, any mutations that decrease M489V gained sensitivity to the ATP-competitive inhibitor Gö6976 autoinhibition of PKCα to promote less constrained or constitu- in cells, with sensitivity to the uncompetitive inhibitor Bis IV tive activation would effectively lead to its degradation, thus par- α remaining similar to that of wild-type PKC . In contrast, the M489V adoxically serving as loss-of-function mutations. Here, we show mutation did not change the IC50 for inhibition by Gö6976 in vitro that one mechanism to evade this is by increasing the reaction rate relative to wild-type protein, revealing that this altered sensitivity is of PKC, in the absence of any perturbations of autoinhibitory specific to the cellular environment. One possible explanation is that constraints (Fig. 7B, Left). The ∼30% increase in the reaction rate the altered dynamics conferred by the M489V mutation now allow of PKCα harboring the M489V germline mutation, accumulating inhibition by ATP-competitive inhibitors, regardless of whether the ’ kinase is bound to protein scaffolds. Alternatively, the altered dy- over a patient s lifetime, could contribute to AD pathogenesis. namics may alter residency time on scaffolds. It is of note that the The results reported here not only reveal how a subtle Met- enhanced sensitivity of PKCα-M489V to the ATP-competitive in- to-Val mutation in a key region of the catalytic domain of α hibitor highlights a weakness that might be pharmacologically PKC can significantly alter molecular dynamics to increase exploited in the treatment of this disease. activity—thus promoting degenerative pathology—but also The amount of PKC activity in the cell exquisitely controls highlight the importance of maintaining exactly the right level cellular homeostasis. This is exemplified by the finding that of PKC signaling, an endeavor that requires precise bio- PKCβII is haploinsufficient in a colon cancer cell line: Two al- chemical and molecular regulation.

Callender et al. PNAS Latest Articles | 7of9 Downloaded by guest on September 24, 2021 Materials and Methods system (Invitrogen). GST-PKCα protein was batch-purified using glutathione Plasmid Constructs, Antibodies, and Reagents. The CKAR was previously de- Sepharose beads as previously described (55) with the following protocol scribed (34, 35). Human PKCα was N-terminally tagged with mCherry via changes: Sf-21 insect cells expressing GST-tagged protein were rinsed with Gateway cloning as described (15, 23). All mutants were generated using PBS and lysed in 50 mM Hepes, pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.1% μ μ QuikChange site-directed mutagenesis (Agilent Genomics). The anti-PKCα Triton X-100, 100 M PMSF, 1 mM DTT, 2 mM benzamidine, 50 g/mL leu- μ antibody (610108) was from BD Transduction Laboratories. The anti-phospho peptin, and 1 M microcystin. Purified protein was exchanged into 20 mM PKCα/βII (pT638/641; 9375S) and pan anti-phosphorylated PKC hydrophobic Hepes, pH 7.5, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT using 10-kDa motif (βII pS660, 9371S) antibodies were from Cell Signaling Technology. The Amicon centrifugal filter unit (EMD Millipore). An equal volume of glycerol − pan anti-phospho-PKC activation loop antibody was previously described (52). was then added to a final concentration of 50% glycerol for storage at 20 °C. The phospho-MARCKS (sc-12971-R) and the total MARCKS (sc-6454) anti- Protein concentration was determined using (i) BSA standards on an SDS/PAGE bodies were purchased from Santa Cruz Biotechnology. β-Actin antibody was gel stained with Coomassie Brilliant Blue stain and (ii)A595 of BGG standards μ μ purchased from Sigma-Aldrich (A2228). PDBu, Gö6976, and Bis IV were from or PKC protein after mixing 5 L of protein solution with 45 L of buffer μ Calbiochem. Lipids used in kinase assays (DG, 800811C and PS, 840034C) were (0.05 M NaOH and 20 mM Tris) and 500 L of Coomassie (Bradford) Protein from Avanti Polar Lipids. Assay Reagent (1856209; Thermo Fisher Scientific).

α Mammalian Cell Culture and Transfection. COS7 cells were maintained in Kinase Assays. The activity of purified GST-PKC (typically 2.4 nM) toward a DMEM (Cellgro) containing 10% FBS (Atlanta Biologicals) and 1% penicillin/ peptide substrate (Ac-FKKSFKL-NH2) was assayed as described previously streptomycin (Gibco) at 37 °C in 5% CO . Transient transfection was carried (56). Standard assay conditions contained (unless otherwise specified in 2 2+ μ 2+ μ out using jetPRIME (PolyPlus Transfection) or FuGENE 6 transfection re- figure legends): Ca (200 M free Ca in the presence of 500 M EGTA), μ μ μ agents (Roche Applied Science) for ∼24 h. 100 M ATP, 100 M substrate, 5 mM MgCl2, 200 M CaCl2, 0.06 mg/mL BSA, and Triton X-100 (0.1% wt/vol) mixed micelles containing 15 mol % PS and PKCα-M489V Mouse Generation. C57BL/6NTac-Prkca mice containing the 5 mol % DG in 50 mM Hepes, pH 7.5, and 1 mM DTT. The quantities of GST- α M489V mutation in Prkca were generated by Taconic Biosciences GmbH for PKC wild type or M489V used in the assay were additionally verified ’ postassay by Western blot using a PKCα antibody. The dependence of PKC Cure Alzheimer s Fund. The point mutation was inserted using a standard 2+ CRISPR/Cas9-mediated gene editing approach. In short, a ribonucleoprotein activity on Ca or mol % PS was analyzed by a nonlinear least squares fit to complex comprising the Cas9 protein complexed with a guide and a trans- a modified Hill equation as described (57) using GraphPad Prism version 6 activator crRNA molecule were injected in fertilized C57BL/6NTac oocytes (GraphPad Software). The Km for peptide substrate or ATP was fit to the – along with a single-stranded oligonucleotide containing the desired point Michaelis Menten equation using GraphPad Prism version 6 (GraphPad 2+ mutation. Insertion of the oligonucleotide sequence resulted in the mutation Software). The concentration of free Ca was calculated using a program 2+ 2+ of the Met 489 to Val and introduction of an AflIII restriction site for geno- that takes into account pH, Ca ,Mg , EGTA, and ATP concentrations typing purposes. Founder mice were genotyped by sequencing and bred to (maxchelator.stanford.edu; CaMgATPEGTA program, ref. 58). C57BL/6NTac wild-type mice for germline transmission. G1 heterozygous mice were identified by restriction analysis and confirmed by sequencing. HET Structure Modeling and Molecular Dynamics Simulations. Homology models of G1 animals were then crossed to C57BL/6NTac wild-type mice for another PKCα wild type and M489V were created using Modeler 9.16 from Protein generation before establishing HET × HET mating to generate cohorts for Data Bank (PDB) ID code 3IW4 (residues 339–597). Molecular dynamics analysis. Twelve- to 15-wk old mice were used for sample preparation. All simulations were performed using GROMACS version 5.0 with the GRO- procedures involving animals were approved by The Scripps Research Insti- MOS96 53a6 force field parameter set. All titratable amino acids were tute’s Institutional Animal Care and Usage Committee (IACUC) and met the assigned their canonical state at physiological pH, short-range interactions guidelines of the National Institute of Health detailed in the Guide for the Care were cut off at 1.4 nm, and long-range electrostatics were calculated using and Use of Laboratory Animals (53). the particle mesh Ewald summation (59). Dispersion correction was applied to energy and pressure terms accounting for truncation of van der Waals Murine Brain Sample Preparation and Immunoblotting. Three-month-old wild- forces and periodic boundary conditions were applied in all directions. Protein type and homozygous M489V mice were killed and hemibrains were obtained constructs were placed in a cubic box of 100 nM NaCl in simple point charge and immediately snap-frozen. Frozen tissue was then homogenized in a dounce water with at least 1 nm distance between the protein construct and box edge tissue grinder with RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, in all directions. Neutralizing counterions were added and steepest decent energy minimization was performed, followed by a two-step NVT/NPT equil- 1% Triton X-100, 1% NaDOC, 0.1% SDS, 10 mM NaF, 1 mM Na3VO4,1mM PMSF, 50 μg/mL leupeptin, 1 μM microcystin, 1 mM DTT, and 2 mM benzamidine). ibration. Both equilibration steps maintained a constant number of particles Homogenates were sonicated and protein was quantified using a BCA protein and temperature, and NVT equilibration was performed for 100 ps main- assay kit (Thermo Fisher Scientific). Thirty micrograms of protein were sepa- taining a constant volume, followed by 10 ns of NPT equilibration maintaining rated by standard SDS/PAGE and transferred to PVDF membranes (BioRad). a constant pressure. Temperature was maintained at 37 °C by coupling protein Membranes were blocked with 5% BSA or 5% milk for 1 h at room temper- and nonprotein atoms to separate temperature coupling baths (60), and ature and analyzed by immunoblotting with specific antibodies. Detection and pressure was maintained at 1.0 bar (weak coupling). All position restraints quantification of immunoreactive bands was conducted by chemiluminescence were then removed and simulations were performed for 400 ns using the on a FluorChem Q imaging system (ProteinSimple). Statistical significance was Nosé–Hoover thermostat (61) and the Parrinello–Rahman barostat (62). Root- determined using a Student’s t test. mean-squared fluctuation (RMSF) analysis compared the SD of the atomic position of each α-carbon in the trajectory, fitting to the starting structure as a FRET Imaging and Analysis. Cells were imaged as described previously (23, 34, 54) reference frame. Images were created using PyMol version 1.7.2.3. with the following modifications: COS7 cells were cotransfected with the indicated mCherry-tagged PKC construct and CKAR (34) at a 0.8:1 ratio of DNA, respectively. ACKNOWLEDGMENTS. We thank Alexandr Kornev, Susan Taylor, Joe Adams, Baseline images were acquired every 15 s for ≥2 min before ligand addition, and and members of the laboratory for many helpful discussions; Alexandr Kornev data were normalized to the baseline FRET ratios. The data are graphed as average for assistance in drafting Fig. 1C; and Amanda Roberts (Animal Models Core Facility, The Scripps Research Institute) for providing the mouse brains. This normalized FRET ratio ± SEM from at least three independent experiments. work was supported by NIH Grants R35 GM122523 and GM43154 (to A.C.N.), the Cure Alzheimer’s Fund (A.C.N.), and Cancer Research UK (J.B.). J.A.C. was Insect Cell Culture and Protein Purification. Human GST-PKCα protein was supported by the University of California, San Diego Graduate Training Pro- expressed and purified from insect cells using the Bac-to-Bac expression gram in Cellular and Molecular Pharmacology (T32 GM007752).

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